A new study by researchers from Shandong Normal University-China and Changchun Veterinary Research Institute-China has discovered that the SARS-CoV-2 open reading frame 10 or ORF10 causes break down of mitochondrial antiviral signaling protein (MAVS), in the process suppressing the human host immune response.
The study team demonstrated that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes.
The mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway.
It was also found that the overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B.
Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10.
The findings reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.
The study findings were published in the peer reviewed journal: Cellular & Molecular Immunology (By Nature). https://www.nature.com/articles/s41423-021-00807-4
The SARS-CoV-2 genome is a positive-sense, nonsegmented, single-stranded RNA with a length of 29.9 kb. The genome of SARS-CoV-2 contains 14 open reading frames (ORFs) flanked by 5′ and 3′ untranslated regions . ORF1a and ORF1b, which account for approximately two-thirds of the viral RNA genome, encode polyproteins that are further cleaved to form 16 nonstructural proteins (nsps) that form the viral replicase-transcriptase complex [6, 7].
The ORFs in the third of the genome near the 3′ terminus encode four main structural proteins, the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, which are components of the virion . In addition, the SARS-CoV-2 genome encodes eleven accessory proteins: ORF3a, ORF3b, ORF3c, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10 .
Although the genome of SARS-CoV-2 (NCBI reference sequence: NC_045512.2)  is similar to that of SARS-CoV (NCBI reference sequence: NC_004718.3) , SARS-CoV-2 exhibits lower pathogenicity and higher infectivity than SARS-CoV .
Compared with the SARS-CoV genome, the SARS-CoV-2 genome uniquely encodes the ORF10 protein, which contains 38 amino acids [13, 14]. ORF10 was reported to bind to an E3 ubiquitin ligase complex containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B, which ubiquitinates host proteins and degrades them via the 26 S proteasome; ZYG11B is dispensable for SARS-CoV-2 infection, and the interaction between ORF10 and ZYG11B is not relevant for SARS-CoV-2 infection . However, how ORF10 functions in the interaction between the virus and host cells is unclear.
SARS-CoV-2 disrupts the host innate immune response via both its structural proteins and Nsps and induces overt but delayed type I interferon (IFN-I) responses . Upon SARS-CoV-2 infection, IFN-I production is decreased, and the release of proinflammatory cytokines is enhanced, disrupting the balance of the immune response, which is responsible for the great severity of SARS-CoV-2 infection .
IFN-I production is rapidly triggered by the recognition of pathogen-associated molecular patterns, such as viral nucleic acids, by host sensors . CoV RNA can be recognized by RIG-I-like receptors (RLRs), including retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation gene 5 (MDA5), in the cytoplasm . After activation, RIG-I and MDA5 can interact with the adapter mitochondrial antiviral signaling protein (MAVS, also termed IPS-1, VISA, and Cardif) [16, 20].
Subsequently, MAVS recruits two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi), leading to activation of the transcription factors interferon regulatory factor 3/7 (IRF3/7) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) .
Then, these proteins trigger signaling cascades to initiate the production of IFNs as well as the downstream expression of multiple IFN-stimulated genes (ISGs) and inflammatory cytokines . The IFN-I response is critical for the response to SARS-CoV-2 infection. The use of IFN combined with ribavirin for the treatment of SARS-CoV infection has shown some beneficial effects [23,24,25].
Furthermore, IFN treatment was found to inhibit SARS-CoV-2 infection in a dose-dependent manner in human Calu-3 cells and simian Vero E6 cells, but the potential of IFN as a therapeutic agent for COVID-19 awaits validation . Recent studies have shown that MDA5, LGP2, and NOD1 are required for the recognition of SARS-CoV-2 by lung epithelial cells and that IRF3, IRF5 and NF-κB/p65 are key transcription factors that regulate the IFN response during SARS-CoV-2 infection .
SARS-CoV-2 has developed multiple strategies to antagonize the innate immune response by attacking these key molecules in the IFN-I signaling pathway. For example, SARS-CoV-2 M targets MAVS and impairs its accumulation and recruitment of downstream signaling components .
SARS-CoV-2 ORF6 antagonizes the host innate immune response by blocking IRF3 activation and STAT1 nuclear translocation . SARS-CoV-2 Nsp14 targets the type I IFN receptor IFNAR1 for lysosomal degradation, thus blocking activation of the crucial transcription factors STAT1 and STAT2 . However, whether SARS-CoV-2 ORF10 plays a role in antagonizing antiviral immunity is unknown.
In the immune system, proper mitochondrial function is a prerequisite for inflammatory responses and host defense . Viral infection can affect the host mitochondrial network, including mitophagy induction . Some viruses trigger mitophagy through different strategies and control the process of mitophagy .
This ability enables viruses to promote persistent infection and attenuate innate immune responses . The study indicates that the matrix protein of HPIV3 induces Parkin-PINK1-independent complete mitophagy, inhibiting host antiviral IFN responses .
In addition, HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by binding to a mitophagy receptor, NIX, on mitochondria and activating NIX-mediated mitophagy to support viral replication . NIX is a typical autophagy receptor that is able to transport mitochondria to autophagosomes through direct interaction with Atg8 family proteins .
NIX-mediated mitophagy is responsible for the elimination of spontaneously aggregated MAVS . Therefore, successful viral infection and replication are largely achieved by the ability of viruses to attenuate the innate antiviral responses mediated by mitochondria . However, there are no relevant studies on the induction of mitophagy by SARS-CoV-2 to escape the host’s innate immune surveillance.
In this study, we demonstrate that SARS-CoV-2 ORF10 inhibits innate immunity and promotes viral replication by inducing mitophagy to degrade MAVS. ORF10 interacts with Nip3-like protein X (NIX) and LC3B and translocates to mitochondria, where it induces mitophagy, leading to MAVS degradation. Further study showed that NIX is involved in the ORF10-mediated degradation of MAVS and blockade of IFN responses.
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To date, at least 26 ORFs have been identified in the SARS-CoV-2 genome but unfortunately many virologist and genomic specialist belong to the ‘old school’ only believe that the five main ORFs (3a, 6, 7a, 7b, 8) are only the key canonical accessory proteins of significance. https://pubmed.ncbi.nlm.nih.gov/32330414/
The only published study showing about 23 ORFs was done by researchers from Israel but a latest non published study shows 26 ORFs in the SARS-CoV-2 genome. https://www.nature.com/articles/s41586-020-2739-1
Many of these ORFs and accessory proteins are believed to play a very important role in the SARS-CoV-2 pathogenesis and ways in affects the cellular systems and pathways of the human host but unfortunately not much attention is being paid to them.
There is a huge variety and diversity of coronaviruses (CoVs) that infect humans, pigs, cattle, horses, rats, and bats, among other host species. These viruses mainly cause infections of the respiratory and digestive tracts, with a wide range of clinical symptoms.
In the past, humans CoVs were thought to be relatively harmless respiratory infections. Zoonotic CoVs, on the other hand, have crossed species boundaries, resulting in novel CoVs that can transfer from animals to humans.
The SARS-CoV that caused SARS outbreaks in 2002 and 2003 and MERS-CoV that emerged in 2012 and continues to circulate in camels are such examples as also the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) that emerged in December 2019 and is responsible for the current severe COVID-19 outbreak worldwide, which has resulted in significant morbidity and mortality.